Investigators are finding ways to target proteins previously thought to be untargetable.

While multiple human cancers are associated with oncogene amplification, epigenetic targets causing amplification such as transcription factors were once considered “undruggable,” or unlikely to be modulated with a small molecule drug. Generally, these proteins lack surface involutions suitable for high-affinity binding by small molecules. But by thinking outside the “loop” or the usual structures required for drug targets, investigators have been making headway in targeting the formerly untargetable.

Multiple human cancers are associated with c-Myc gene amplification including lung carcinoma breast carcinoma, colon carcinoma, and neuroblastoma. The protogene also plays a key role in cell cycle regulation, metabolism, apoptosis, differentiation, cell adhesion, as well as in tumorigenesis, and participates in regulating hematopoietic homeostasis. Its gene product functions as a transcription regulator, part of an extensive network of interacting factors regulating the expression, it has been estimated, of more than 15 percent of all human genes.

While Myc oncogene family members, for example, act as key drivers in human cancers, they have been considered undruggable as they encode transcription factors and carry out essential functions in proliferative tissues, suggesting that their inhibition could cause severe side effects. And from a chemist’s point of view, these proteins’ surfaces are not amenable to binding drugs. In an online dialog posted on the NCI’s website in October of 2010, an investigator noted, “We don’t know how to interfere with these factors or their activities in clinical settings because, in general, we lack the means to inhibit proteins that are not enzymes.”

But by preventing key protein-protein interactions that enable the actions of these transcriptional drivers, scientists are drugging the formerly undruggable.

To Drug the Undruggable Target

One such approach was reported in Nature in 2009 as a team of Harvard scientists said that they had successfully targeted a “master” protein, Notch1, which had been considered “untouchable” by conventional drugs. The protein is a key transcription factor regulating genes involved in cell growth and survival but like other transcription factors has proven an elusive drug target due to its structure. The scientists said they had designed a synthetic, cell-permeable alpha-helical peptide, SAHM1, which could target a critical protein-protein interface in the notch transactivation complex. The drug molecule enters cells and interferes with a protein-protein interaction essential for the transmission of cell growth signals via the Notch pathway.

The researchers tested the drug using cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) and a mouse model of the disease. The Notch1 gene is mutated in half of patients with T-ALL and produces an inappropriately active Notch1 protein. Activated Notch signaling has been seen in several other cancers including lung, ovarian, and pancreatic cancer, and melanoma.

“We’ve drugged a so-called undruggable target,” said Gregory L. Verdine, Ph.D., Erving professor of chemistry at Harvard University. “This study validates the notion that you can target a transcription factor by choosing a new class of molecules, namely stapled peptides.” He added that, because the molecular logic of these proteins is similar to Notch1’s, this strategy might work for other transcription factors as well.

Targeting BET

Another emerging approach to drugging the undruggable is to target the bromo and extra C-terminal domain (BET) family of bromodomains that are involved in binding epigenetic “marks” on histone proteins. Four members of this 47-protein family interact with chromatin including histone acetylases and nucleosome remodeling complexes. Bromodomain proteins act as chromatin “readers” to recruit chromatin-regulating enzymes, including “writers” and “erasers” of histone modification, to target promoters and to regulate gene expression. As mentioned in a previous GEN article, epigenetic control systems generally involve three types of proteins: “writers”, “readers”, and “erasers.” Writers attach chemical marks, such as methyl groups (to DNA) or acetyl groups (to the histone proteins that DNA wraps around). Readers bind to these marks, thereby influencing gene expression. Erasers remove the marks.

While investigators have considered that the precise function of the so-called BET bromodomain remains incompletely defined, proteins containing this domain have become another epigenetic target for drug development companies. Importantly, these domains may allow researchers a way to get at oncogenic targets that were once thought undruggable including the proto-oncogene Myc.

Small molecule inhibition of BET protein bromodomains also selectively suppresses other genes such as Bcl-2 that have important roles in cancer, as well as some NF-κB-dependent genes that have roles in both cancer and inflammation. Small molecule inhibition of BET bromodomains leads to selective killing of tumor cells across a range of hematologic malignancies and in subsets of solid tumors. In particular, the bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt’s lymphoma, human nuclear protein in testis (NUT) midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer.

BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function, until very recently, remained unknown.

In 2010, investigators reported in Nature that they had identified a cell-permeable small molecule that bound competitively to bromodomains, or acetyl-lysine recognition motifs. Competitive binding by the small molecule JQ1, the investigators reported, displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models.

The authors say that these data established proof-of-concept for targeting protein–protein interactions of epigenetic readers, and could provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

More recently, writing in the Journal of Medicinal Chemistry, investigators at GlaxoSmithKline reported that they had successfully optimized a class of benzodiazepines as BET bromodomain inhibitors, apparently without any prior knowledge of identified molecular targets. Significant medicinal chemistry provided the bromodomain inhibitor, I-BET762 or GSK525762, which is currently in a Phase I clinical trial for the treatment of NUT midline carcinoma, a rare but lethal form of cancer, and other cancers.

Casting a Wide Net

Constellation Pharmaceuticals of Cambridge, MA, announced that it has initiated a Phase I clinical trial of CPI-0610, a novel small molecule BET protein bromodomain inhibitor, in patients with previously treated and progressive lymphomas. This first-in-human trial is currently open at Sarah Cannon Research Institute in Nashville, Tennessee, and at the John Theurer Cancer Center in Hackensack, New Jersey. Additional study sites in the U.S. will join the trial over the next several months. Studies of CPI-0610 are also planned in patients with multiple myeloma and in patients with acute leukemia or myelodysplastic syndrome.

Constellation’s CMO, Michael Cooper, M.D. told GEN that “small molecule inhibitors of BET protein bromodomains have demonstrated broad activity against hematologic malignancies in preclinical models. And this activity can be achieved in vivo with levels of compound exposure that are well tolerated. While we are encouraged by these observations, what really makes the area interesting is the novel mechanism by which BET protein bromodomain inhibitors elicit their biologic effects. They disrupt the interaction of BET proteins with acetylated lysine residues on histones and thereby suppress the transcription of key cancer-related genes such as MYC, BCL-2, and a subset of NF-κB-dependent genes. These genes have in the past been difficult to target with small molecules. In light of the breadth of the activity in preclinical models of hematologic malignancies and the important genes that are targeted, we intend to cast a wide net across hematologic malignancies in the clinic.”

Robert Sims, Ph.D., and senior director of biology at Constellation explained that BET protein bromodomain inhibition is only of several areas of interest for the company. “The BET proteins constitute one class of epigenetic targets, namely molecules that recognize patterns in chromatin architecture and either enhance or suppress gene transcription. Constellation’s approach to epigenetics also includes programs in the enzymes that modify the architecture of chromatin, for example by the methylation or demethylation of histone proteins (writers and erasers, respectively). Even though our first drug candidate is directed against a set of reader proteins, we are also looking at inhibitors of the writer protein, EZH2, which is mutated in some types of non-Hodgkin lymphoma and overexpressed in many malignancies.”

In January 2012, Constellation and Genentech announced collaboration based on the science of epigenetics and chromatin biology to discover and develop innovative treatments for cancer and other diseases. Each company will each commit a significant portion of their research and development efforts to the advancement of programs under the collaboration, and each party will have the right to retain exclusive rights to programs emerging from the collaboration.

And more biotech giants can be expected to enter the field of epigenetics as smaller companies advance into the clinic with this novel approach to controlling gene expression gone wrong in cancer cells.